The role of oestrogen receptor a in human thyroid cancer: contributions from coregulatory proteins and the tyrosine kinase receptor HER2

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1 Endocrine-Related Cancer (2) The role of oestrogen receptor a in human thyroid cancer: contributions from coregulatory proteins and the tyrosine kinase receptor HER2 Dara O Kavanagh, Marie McIlroy,2, Eddie Myers, Fiona Bane 2, Thomas B Crotty, E McDermott, Arnold D Hill,2 and Leonie S Young,2 School of Medicine and Medical Science, UCD Conway Institute, St Vincent s University Hospital and University College Dublin, Dublin 4, Ireland 2 Endocrine Oncology Research Group, Department of Surgery, Royal College of Surgeons in Ireland, St Stephens Green, Dublin 2, Ireland (Correspondence should be addressed to L S Young at Endocrine Oncology Research Group, Department of Surgery, Royal College of Surgeons in Ireland; lyoung@rcsi.ie) Abstract Epidemiological, clinical, and molecular studies suggest a role for oestrogen in thyroid cancer. How oestrogen mediates its effects and the consequence of it on clinical outcome has not been fully elucidated. The participation of coregulatory proteins in modulating oestrogen receptor (ER) function and input of crosstalk with the tyrosine kinase receptor HER2 was investigated. Oestrogen induced cell proliferation in the follicular thyroid cancer (FTC)-33 cells, but not in the anaplastic 835C cell line. Knockdown of the coactivator steroid receptor coactivator (SRC)- inhibited FTC-33 basal, but not oestrogen induced, cell proliferation. Oestrogen also increased protein expression of and the ER target gene cyclin D in the FTC-33 cell line. ERa,ERb, the coregulatory proteins and nuclear corepressor (), and the tyrosine kinase receptor HER2 were localised by immunohistochemistry and immnofluorescence in paraffinembedded tissue from thyroid tumour patients (nz). ERa was colocalised with both and to the nuclei of the tumour epithelial cells. Expression of ERa and was found predominantly in non-anaplastic tumours and was significantly associated with well-differentiated tumours and reduced incidence of disease recurrence. In non-anaplastic tumours, HER2 was significantly associated with, and these proteins were associated with poorly differentiated tumours, capsular invasion and disease recurrence. Totally, 87% of anaplastic tumours were positive for. Kaplan Meier estimates of disease-free survival indicated that in thyroid cancer, strongly correlates with reduced disease-free survival (P!.), whereas predicted increased survival (P!.). These data suggest opposing roles for the coregulators and in thyroid tumour progression. Endocrine-Related Cancer (2) Introduction Thyroid carcinoma constitutes % of all new malignant disease. Of these, 94% are differentiated follicular or papillary carcinomas. A further 5% are medullary carcinomas derived from neuroendocrine cells. The remaining are anaplastic tumours arising from dedifferentiation of the differentiated type (Figge 999). In view of the low incidence and largely favourable prognosis, therapeutic advances are minimal. Despite multimodal therapy, there has been no improvement in survival rates over the past two decades (Teppo et al. 998, Sherman 23). Malignant disease of the thyroid gland is three times more common in females than in males. Despite this, the prognosis is more favourable in females. Welldifferentiated carcinomas have a female preponderance Endocrine-Related Cancer (2) //7 255 q 2 Society for Endocrinology Printed in Great Britain DOI:.677/ERC-9-26 Online version via

2 D O Kavanagh et al.: Oestrogen in human thyroid cancer and are most frequent in the postpubertal and premenopausal age groups. The use of the oral contraceptive pill is associated with a higher risk of thyroid cancer. An increased incidence is similarly seen in patients treated with oestrogen therapy for gynaecological conditions, but not for postmenopausal patients treated with low-dose oestrogen replacement therapy. Recently, it has been shown that among parous women of reproductive age, a recent pregnancy is associated with approximately a doubling in thyroid cancer risk. Pregnancy is associated with elevated serum thyroid hormone and oestrogen level, further supporting a role for oestrogen in thyroid carcinogenesis (Rossing et al. 2). This gender difference is observed worldwide and suggests that thyroid tumour development and progression may be influenced by oestrogen as previously demonstrated in breast cancer. Furthermore, Kishino et al. (997) identified a beneficial role for high-dose tamoxifen in multidrug resistant thyroid cancer. Oestrogens play a critical role in endocrine tumours, including those of the breast, prostate and thyroid. Oestrogen mediates its genomic actions through binding its nuclear receptor leading to transcription and translation of genes relevant to tumour progression. The oestrogen receptor (ER) is encoded for by two genes, ERa and ERb. Though both isoforms of the receptor have been identified in human thyroid tumour tissue, it is ERa that has been associated with increased oestrogen-dependent cell proliferation (Zeng et al. 28). Oestrogen can also mediate its effects independently of its classic nuclear receptor. Studies in the thyroid have shown that oestrogen can utilise the G-coupled protein GPCR3 to drive both gene transcription and cellular growth (Vivacqua et al. 26). Central to the functioning of ER are the coregulatory proteins. These are present at rate-limiting amounts in the nucleus, thereby regulating transcription of target genes. Coactivators possess histone acetylation activity, which may directly influence gene expression through local depression of chromatin. These coactivators include the p6 (6 kda) protein family members steroid receptor coactivator () and amplified in breast cancer (AIB-). In the absence of ligand, the ER maintains transcriptional silencing through recruitment of the nuclear corepressors (s) silencing mediator of retinoid and thyroid receptors and (Chen & Evans 995, Jenster & Spencer 997). On binding to its receptor, oestrogen releases the resident corepressor and recruits coactivator in order to initiate successful transcription and translation of the target gene (Smith & O Malley 24). In the setting of breast cancer, these molecular switches have been shown to be prognostically significant (Myers et al. 24, McIlroy et al. 26). Aberrant expression of p6 proteins has been associated with resistance to endocrine therapies and the development of tumour recurrence (Osborne et al. 23, Redmond et al. 29). Furthermore, unlike other oncogenes, recent studies provide evidence of a specific role for in the development of metastasis (Qin et al. 29, Wang et al. 29). The presence of the coregulatory proteins in thyroid cancer and their prognostic significance, if any, have not yet been examined. We hypothesised that ER signalling plays a role in the progression of thyroid cancer. To test this, we examined the proliferative effects of oestrogen in thyroid cancer and the effects of oestrogen on the expression of its target gene, cyclin D. In addition, we analysed a cohort of thyroid cancer patients to determine the presence and significance of ERa, ERb and the coregulatory proteins, and, and the tyrosine kinase receptor HER2 in relation tumour type and disease progression. Materials and methods Patient selection Tumour specimens from consecutive patients with thyroid cancer treated at St Vincent s University Hospital, Dublin from 99 to 2 were selected. Histologically, normal thyroid tissue specimens were obtained from patients who underwent surgery for multinodular goitre. The cancer specimens arose from patients who had not had previous therapy for thyroid cancer and had undergone primary surgical resection. Patients with non-anaplastic tumours! mm underwent surgery alone, and those with tumours O mm underwent surgery followed by radio-iodine therapy. Patients with anaplastic tumours received radio-therapy. Clinicopathological parameters Variables analysed included size, pathological subtype, extremes of age (! and O4), gender, degree of differentiation and capsular invasion. Median follow-up was.2 years. Immunohistochemistry Five micron (5 mm thick) tissue sections were taken from paraffin-embedded thyroid cancer and multinodular goitre. Following antigen retrieval, sections were incubated with primary antibodies as 256

3 Endocrine-Related Cancer (2) follows: rabbit anti-human ERa ( mg/ml), rabbit anti-human ( mg/ml), rabbit anti-human ( mg/ml; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-human ERb (5 mg/ml; Serotec, Oxford, UK). The primary antibodies were incubated for h at room temperature. Sections were subsequently incubated with the corresponding biotinlabelled secondary antibody (.5% in PBS; Vector Laboratories, Burlingame, CA, USA) for 3 min, followed by peroxidase-labelled avidin biotin complex (Vector Laboratories) for 3 min. Sections were developed in 3,3-diaminobenzidine tetrahydrochloride for 8 min and counterstained with haematoxylin for 3 min, then passed through increasing concentrations of industrial methylated spirits (IMS) (7 and %) and then xylene. Immunostained slides were scored using the Allred scoring system (Harvey et al. 999). A combined score of three or higher was defined as positive staining. Each slide was observed by two independent observers blinded to the clinicopathological factors of interest. The interobserver correlation coefficient was determined. These coefficients ranged from.99 to.972 indicating a high level of interobserver reliability (Fleiss 986). Assessment of HER2 status HER2 status was evaluated using the Dako (Glostrup, Denmark) HercepTest immunocytochemical assay. Scoring was assessed according to the manufacturer s instructions ( 3). In tumour samples scoring C2 with the Hercept test, HER2 status was confirmed by fluorescent in situ hybridisation using the PathVysion kit probe to detect amplification of the HER2 gene (spectrum orange labelled HER2 and spectrum green labelled a satellite centromeric region for chromosome 7; Vysis Inc., Downers Grove, IL, USA) according to the manufacturer s instructions. Immunofluoresence Thyroid cancer sections were prepared as above and incubated in goat serum (ERa) for 6 min. Goat antirabbit ERa ( mg/ml in % human serum) was placed on each slide for 9 min. The sections were rinsed in PBS and incubated with the corresponding secondary fluorochrome-conjugated antibody ( in ; Sigma Aldrich) for 6 min. The slides were rinsed in PBS and blocked in rabbit serum for 9 min and washed with PBS. Each slide was incubated with either rabbit antihuman or rabbit anti-human (both mg/ml in % human serum) for 9 min, followed by a wash in PBS. The slides were incubated with the corresponding fluorochrome-conjugated antibody ( in ) for 6 min. Sections were rinsed in PBS and mounted (Dako). Sections were examined under a fluorescent microscope. Negative controls were performed using matched IgG, and no staining was detected. Cell culture treatments The follicular thyroid cancer (FTC)-33 cell line (ECACC, Wiltshire, UK) was grown in DMEM and Ham s F2 (:) supplemented with % FCS and 2mM L-glutamine (Gibco). The 835C anaplastic thyroid cancer cell line 835C (ECACC) was grown in EMEM (Sigma) supplemented with % FCS, 2 mm L-glutamine and % non-essential amino acids. Cells were incubated in a humidified atmosphere of 5% CO 2 at 37 8C. Experiments were carried out when cells reached 9% confluence. Cells were maintained in steroid and phenol-free minimum essential medium (Gibco) for 72 h prior to treatment. Cells were then incubated in the presence and absence of 7b-oestradiol (OE 2 ) or Faslodex (ICI 82 78) ( nm) for 24 h and then harvested. Western blotting Proteins ( mg) were resolved on a polyacrylamide gel (2% for ERa, and cyclin D, and 6% for ) at V for 2 min and were transferred to a nitrocellulose membrane (25 ma for 6 min for ERa, and cyclin D, and 9 min for ). Membranes were incubated for 6 min in blocking buffer (5% non-fat dry milk and.5% Tween-2 in TBS) at room temperature and subsequently with primary antibody, rabbit anti-human ERa (2 mg/ml), rabbit anti-human ERb (2 mg/ml), rabbit anti-human (2 mg/ml), rabbit anti-human (2 mg/ml) or rabbit anti-human cyclin D (2 mg/ml) in blocking buffer overnight at 4 8C. The membranes were washed prior to incubation with the corresponding HRP secondary antibody ( in 2 for ERa, ERb, cyclin D and, and in 33 for ) in blocking buffer for 6 min at room temperature. The membranes were washed and developed with intensified chemiluminescence (Pierce, Rockford, IL, USA). Proliferation assays The FTC-33 and 835C cells were seeded on a 2-well plate. Cells were treated with either nm 7-OE 2 or ICI alone or in combination as described above. Methyl thioazole tetrazolium (5 mg/ml) was added to each well, and following a 4-h incubation period, absorbance was read at 57 nm

4 D O Kavanagh et al.: Oestrogen in human thyroid cancer sirna Pre-designed and validated sirna directed against (Ambion, Austin, TX, USA, cat no. 5458) were used in the knockdown studies (Redmond et al. 29). Flow cytometry Cells were harvested by centrifugation at 2 g for 4 min and washed in ml ice-cold PBS. Cell pellets were resuspended in 2 ml PBS and fixed in 2 ml ice-cold 7% (v/v) ethanol at 4 8C overnight. Cells were centrifuged at 3 g for 4 min, ethanol was removed and the pellets were resuspended in 4 ml PBS. RNase A (.5 mg/ml) and propidium iodide (2 mm) were added, and samples were incubated in the dark at 37 8C for 3 min. Cell cycle profiles were analysed on an Accuri C6 flow cytometer using CFlow Software. Statistical analysis Univariate analysis was performed using Fisher s exact test for categorical variables and using Wilcoxon s test for continuous variables. Two-sided P values of!.5 were considered to be statistically significant. Kaplan Meier estimates of disease-specific survival functions were computed, and the Wilcoxon test was used to compare survival curves. Results Thyroid cancer cell cycle, proliferation and protein expression Cell cycle phase of follicular (FTC-33) and anaplastic (835C) thyroid cancer cell lines was assessed by flow cytometry. Anaplastic thyroid cancer cells had a greater percentage of cells in the G 2 /M phase in comparison to the follicular cell line (Fig. A). There was no discernable difference observed in cell cycle phase in either of the thyroid cancer cells following treatment with b-oe 2. However, oestrogen increased overall cellular proliferation of the FTC-33 cell line (Fig. B). The ER antagonist ICI (Faslodex) inhibited oestrogen-dependent FTC-33 cell proliferation. Oestrogen had no effect on cell proliferation in the 835C cell line. Treatment with oestrogen increased protein expression of the ER coactivator and its target gene cyclin D in the FTC-33 cell line (Fig. C) and decreased expression of ERb, with no alterations in ERa, or HER2 protein expression detected (Fig. C). In contrast, in the 835C cells, oestrogen failed to significantly alter protein expression of ER, its target gene cyclin D or HER2 (Fig. C). Levels of and were found to be low in the 835C cell line. Knockdown of significantly retarded basal cell proliferation in the FTC-33 cells, but had no effect on oestrogen-induced cell proliferation or on proliferation in the 835C cell line (Fig. D). Localisation of ER and coregulatory proteins in human thyroid cancer tissue ERa, ERb, and were all found to be expressed in a subset of thyroid tumour patients. ERa and the coregulatory proteins were strongly expressed in the nucleus with some cytoplasmic staining also observed (Fig. 2A). Expression of was detected exclusively in the tumour tissue, whereas ERa and were also present in the surrounding normal and goitre tissue. In patients who were positive for ERa and the coregulatory proteins, ERa was found to colocalise with both and in the nucleus of the tumour epithelial cells (Fig. 2B). Expression of ER and coregulatory proteins in thyroid tumour subtypes Qualitative expression of the receptors ERa and HER2, along with the coregulatory proteins and, was examined in subtypes of thyroid cancers (Fig. 3A). ERa and were found to be expressed in both normal thyroid tissue and non-anaplastic tumours, including papillary, follicular and adenoma. ERb was found to be expressed predominantly in papillary tumours. A low percentage (!%) of papillary and follicular tumours expressed. In contrast, a high percentage of anaplastic tumours (87%) expressed, and a low number of these tumours were positive for ERa. In the patient population, ERa expression was positively associated with and negatively associated with HER2 and (Table ). In terms of clinicopathological characteristics, no association was observed between expression of the ERa and the coregulatory proteins in relation to extremes of age (! and O4 years), gender or tumour size. ERa and expression was positively associated with well-differentiated tumours and inversely with disease recurrence. Whereas, both the tyrosine kinase receptor HER2 and coactivator associated with capsular invasion and recurrence (Table ). Kaplan Meier estimates of disease-free survival indicate that patients with anaplastic tumours have significantly reduced survival compared with 258

5 Endocrine-Related Cancer (2) A FTC-33 P < Control Oestradiol Faslodex Oestradiol and faslodex % cells in G2/M phase 835C Control Oestradiol Faslodex Oestradiol and faslodex Count FL2-A Count FL2-A B Proliferation index (IP).5.5 FTC-33 2 P <. Control Oestradiol Faslodex Oestradiol and faslodex Proliferation index (IP) C Control Oestradiol Faslodex Oestradiol and faslodex C ERα FTC C ERβ Cyclin D HER2 β-actin Control Oestradiol Control Oestradiol Relative protein expression 2.5 P <. P < C E C E C E C E C E C E ERα ERβ Cyclin D HER2 Relative protein expression C E C E C E C E C E C E ERα ERβ Cyclin D HER2 D Scr sirna FTC-33 sirna Scr sirna 835C sirna FTC-33 No treatment P = Scrambled sirna- Proliferative index Proliferative index Estrogen treatment Scrambled E2 sirna Control Oestradiol Control Oestradiol 835C Proliferative index.2 Scrambled sirna- E Scrambled E2 sirna- E 2 Proliferative index Figure (A) Flow cytometry analysis of cell cycle phase in follicular (FTC-33) and anaplastic (835C) breast cancer cell lines. Percentage of cells in follicular (FTC-33) and anaplastic (835C) thyroid cancer cell lines. Percentage of cells in G 2 /M phase under control conditions and following treatment with 7b-oestradiol and Faslodex (48 h) alone and in combination. Results are expressed as meangs.d. of individual experiments (nz3). (B) Cell proliferation was analysed by MTT assay. FTC-33 and 835C thyroid cancer cells were cultured in the presence of 7b-oestradiol and Faslodex (24 h) alone and in combination. Results are expressed as meangs.d. of individual experiments (nz3). (C) Protein expression of ERa, ERb,, and cyclin D in the FTC-33 and 835C human thyroid cancer cell line was assessed by western blotting post-incubation with b-oestradiol (E) (24 h). Membranes were also probed for b-actin. Optical density readings were calculated relative to b-actin, control values (C) were normalised to one and the treated group was expressed as a ratio. Results are expressed as meangs.d. (nz3). (D) Successful knockdown of with sirna was confirmed by western blot. Knockdown of inhibited basal cell proliferation in the follicular (FTC-33), but not anaplastic (835C) thyroid cancer cell line. Results are expressed as meangs.d. (nz3)

6 D O Kavanagh et al.: Oestrogen in human thyroid cancer A Thyroid cancer IgG-matched control Normal thyroid ERα B ERα ERα + ERα ERα + Figure 2 (A) Immunohistochemical localisation of ERa, and counterstained with haematoxylin and matched IgG-negative controls in human thyroid cancer (2!) and normal thyroid (2!). (B) Immunofluorescent colocalisation of ERa with and in human thyroid cancer. non-anaplastic tumours (P!.; Fig. 3B). In the whole patient cohort, significantly predicted poor disease-free survival (P!.), whereas expression of predicted a good prognosis (P!.; Fig. 3B). In order to more clearly define a role for these coregulatory proteins in thyroid cancer subtypes, we analysed expression of, and HER2 expression in anaplastic versus non-anaplastic tumours (Table 2). was expressed in 55% of nonanaplastic tumours and associated positively with well-differentiated cancers (P!.). Both and HER2 were expressed in 7% of non-anaplastic tumours, and these proteins were significantly associated with each other (P!.) and with disease recurrence (PZ. and P!.5 respectively). Totally, 87% of anaplastic tumours were positive for, whereas no expression of either or HER2 was observed. All patients with anaplastic tumours had a disease recurrence (Table 2). Discussion Epidemiological, translational and clinical evidence suggests a role for oestrogens in the development of thyroid cancer (Kishino et al. 997, Rossing et al. 2, Lee et al. 23, Zeng et al. 28). Recent reports suggest that oestrogen can increase ERa expression in non-anaplastic papillary cancer cells, increase cellular proliferation and inhibit pro-apoptotic protein expression. In thyroid cancer, oestrogen can also activate extranuclear effects of ER, in particular by signalling through the G protein-coupled receptor GPR3 (Vivacqua et al. 26). No alteration, however, 26

7 Endocrine-Related Cancer (2) A Percentage expression ERα ERβ HER2 Papillary Follicular Adenoma Anaplastic Normal B (i) Anaplastic Kaplan Meier survival estimates anaplastic vs. all other pathologies P <. All others 8 3 Anaplastic 5 5 Analysis time (ii) Kaplan Meier survival estimates impact of ve P <. +ve Analysis time (iii) Kaplan Meier survival estimates impact of P =.38 ve +ve Analysis time Figure 3 (A) Expression levels of ERa, ERb,, and HER2 in thyroid cancer specimens represented as percentages observed in each of the pathological subtypes encountered. (B) Patients with anaplastic disease were found to have a significantly reduced period of disease-free survival (i). Positivity for was shown to result in poor survival (ii), whereas expression of associates with longer disease remission (iii). in cellular proliferation in anaplastic thyroid carcinoma cells has been observed. In this study, we employed cell line models of follicular and anaplastic thyroid cancer. The follicular cancer cells (FTC-33) were originally derived from a lymph node metastasis of a follicular thyroid carcinoma. These cells do, however, retain differentiated thyrocytic function and are responsive to growth factors. The anaplastic cell line, 835C, was established from an undifferentiated thyroid and is positive for both EGFR and HER2 (Murakawa et al. 25). From cell cycle analysis, 835C cells had a greater percentage of cells in the G 2 /M phase in comparison to the FTC-33 cells. oestrogen had no effect on cell cycle phase in either cell line. However, in the FTC-33 cells, oestrogen induced cellular proliferation, which was inhibited by the ER antagonist ICI (Faslodex) and increased protein expression of its target gene cyclin D. In our patient cohort, ERa was found to be highly expressed in both normal thyroid tissue and non-anaplastic tumours, including papillary, follicular and adenoma. However, only % of anaplastic tumours were positive for ERa. Expression of the steroid receptor was positively associated with well-differentiated tumours and 26

8 D O Kavanagh et al.: Oestrogen in human thyroid cancer Table Associations with clinicopathological parameters as determined by Fisher s exact test. Association of variables oestrogen receptor a (ERa), steroid receptor coactivator- (), nuclear corepressor (), HER2 and recurrence were evaluated in conjunction with clinical classifications Total ERa positive (nz63) positive (nz25) positive (nz57) HER2 positive (nz8) Recurrence (nz27) Capsular invasion (P value) nz24 9/63 (.28) a 3/25 (!.) 5/57 (Z.) a 7/8 (!.) 7/27 (.593) Well differentiated (P value) nz76 54/63 (!.) /25 (!.) a 52/57 (!.) /8 (!.) a 7/27 (!.) a ERa positive nz63 5/25 (!.) a 42/57 (!.) 5/8 (Z.9) /27 (.25) a positive (P value) nz25 5/63 (!.) a /57 (!.) a /8 (!.) 6/27 (!.) NcoR positive (P value) nz57 42/63 (!.) /25 (!.) a 2/8 (!.) a 8/27 (Z.4) a HER2 positive (P value) nz8 5/63 (.7) a /25 (!.) 2/57 (!.) a 8/27 (.39) Recurrence nz27 4/63 (.3) a 2/25 (!.) 2/57 (!.) a 7/8 (.7) a Reverse relationship. inversely with disease recurrence. These data suggest that in thyroid cancer, oestrogen signalling is associated with non-aggressive, well-differentiated tumours, which have a favourable prognosis. Where the proliferation of thyroid cancer cells is promoted by ERa, proliferation is thought to be reduced by enhanced expression of ERb (Zeng et al. 28). In this study, ERb expression was observed in both the follicular and anaplastic cancer cell lines; however, at a tissue level, ERb was found to be expressed predominantly in papillary thyroid cancer patients, suggesting that a functional role for ERb may be principally in this cancer subtype. The magnitude of ER gene regulation is influenced not only by the ligand, but also by the presence of specific co-regulatory proteins, present at rate-limiting levels, which modulate transcription. Studies from our group in breast cancer suggest that while expression of the coactivator protein correlates with reduced time to disease recurrence, the presence of the corepressor predicts enhanced disease-free survival (Myers et al. 25, Al-azawi et al. 28, Redmond et al. 29). In the thyroid patient population, was found exclusively in the non-anaplastic tumours and normal thyroid tissue. The corepressor significantly associated with expression of ERa, inversely associated with capsular invasion and positively with well-differentiated tumours. Furthermore, Kaplan Meier estimates of disease-free survival demonstrated that significantly predicted enhanced survival in thyroid cancer patients. In contrast, in the FTC-33 follicular cancer cell line, protein expression was elevated in the presence of oestrogen, and knockdown of the coactivator protein inhibited cell proliferation. However, knockdown of had no effect on oestrogen-induced FTC-33 cell growth, suggesting that coactivator functional redundancy similar to that seen in the breast may also be relevant in thyroid cancer (Xu & Li 23). was expressed in a subset of non-anaplastic patients. Expression of was associated with disease recurrence and inversely associated with well-differentiated tumours. Though only low levels of were detected in the 835C anaplastic cell line and knockdown of the coactivator had no effect on cell proliferation, was found to be highly expressed in the anaplastic patient population. These observations are in line with recent studies in the breast, which describe a specific role for in the development of tumour metastasis (Qin et al. 29, Wang et al. 29). Furthermore, in the entire thyroid cancer patient population, there was a Table 2 Association of steroid receptor coactivator- (), nuclear corepressor (), HER2, differentiation and recurrence were evaluated by Fisher s exact test in the anaplastic and non-anaplastic patient populations Anaplastic (nz8) Non-anaplastic (nz3) (nz7) HER2 (nz) (nz) (nz8) HER2 (nz8) (nz57) 7/8 P!. /57 P!.* HER2 /7 /8 P!. 2/57 P!.* /7 /8 P!.* 2/8 P!.* Recurrence 7/7 9/8 PZ. 8/8 P!.5 8/57 PZ.24 Well differentiated /7 /8 P!.* /8 P!.* 52/57 P!. *Inverse relationship

9 Endocrine-Related Cancer (2) strong correlation between expression and reduced disease-free survival. Abnormalities in growth factor signalling pathways play an intrinsic role in endocrine tumour disease progression. In human breast cancer, the growth factor receptor HER2 is overexpressed in 2 3% of tumours (Berger et al. 988). Molecular and clinical evidence suggests that crosstalk between steroid receptor and growth factor pathways contributes to endocrine insensitivity, at least in part through phosphorylation and activation of coactivator proteins (Osborne et al. 25). We have previously described a positive association between expression of the coactivators, and AIB, and the growth factor receptor, HER2, in a cohort of breast tumour patients (Myers et al. 25) and shown that the risk ratio of recurrence in HER2-positive patients with elevated is 6.82 (Fleming et al. 24). Several groups have reported HER2 expression in thyroid cancer (Ensinger et al. 23, Mondi et al. 23, Wiseman et al. 28), though results from these studies remain inconclusive. Mondi et al. (23) observed no significant expression of HER2 in benign or malignant thyroid tissue, and Wiseman et al. (28) found that HER2 was not significantly expressed in anaplastic tumours. However, others have reported that in papillary thyroid carcinoma, expression of HER2 was associated with disease recurrence (Ensinger et al. 23). In this study, though no detectable levels of HER2 were observed in anaplastic tumours, 7% of non-anaplastic tumours were positive for HER2. Expression of the tyrosine kinase receptor was associated with capsular invasion, inversely with well-differentiated tumours and positively with disease recurrence. Of interest, in the non-anaplastic tumour population, HER2 was positively associated with and inversely associated with. This is the first translational study to take a comprehensive look at oestrogen signalling in relation to the contribution of steroid receptor coregulatory proteins and tyrosine kinase receptor status in human thyroid cancer. Data that have emerged from this study establish ER signalling, in conjunction with its corepressor protein as a mediator of welldifferentiated tumours with a favourable prognosis. The coactivator protein is associated with invasion, poor differentiation, tumour recurrence and reduced disease-free survival. In non-anaplastic tumours, strongly associates with HER2, suggesting that crosstalk with the tyrosine kinase receptor may activate in this tumour subtype. In anaplastic thyroid cancer, does not appear to mediate these effects through the steroid receptor ER. The ability of steroid coactivator proteins to function independently of ER has been previously described by our group and others (Goel & Janknecht 24, Myers et al. 25, Al-azawi et al. 28). Though the signalling mechanism of in cellular dedifferentiation in anaplastic thyroid carcinoma has yet to be resolved, this protein may represent a new therapeutic target for this rare, but rapidly fatal disease. Declaration of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Funding This work was supported by Breast Cancer Ireland (grant number 6/92, 26). Author contribution statement D Kavanagh, E Myers, M McIlroy and F Bane performed the experiments; M McIlroy was responsible for statistical analysis and manuscript preparation; T Crotty, pathology expertise; E McDermott and A Hill provided tumour specimens and were instrumental in creating a clinical framework to relate the data back to the patient; Leonie Young designed the experiments and was responsible for the successful execution of the study. References Al-azawi D, Ilroy MM, Kelly G, Redmond AM, Bane FT, Cocchiglia S, Hill AD & Young LS 28 Ets-2 and p6 proteins collaborate to regulate c-myc in endocrine resistant breast cancer. Oncogene Berger MS, Locher GW, Saurer S, Gullick WJ, Waterfield MD, Groner B & Hynes NE 988 Correlation of c-erbb-2 gene amplification and protein expression in human breast carcinoma with nodal status and nuclear grading. Cancer Research Chen JD & Evans RM 995 A transcriptional corepressor that interacts with nuclear hormone receptors. Nature Egawa C, Miyoshi Y, Iwao K, Shiba E & Noguchi S 2 Quantitative analysis of estrogen receptor -alpha and -beta messenger RNA expression in normal and malignant thyroid tissues by real-time polymerase chain reaction. Oncology Ensinger C, Prommegger R, Kendler D, Gabriel M, Spizzo G, Mikuz G & Kremser R 23 Her2/neu expression in poorly-differentiated and anaplastic thyroid carcinomas. Anticancer Research Figge J 999 Epidemiology of thyroid cancer. In Thyroid Cancer: a Comprehensive Guide to Clinical Management, pp Ed L Wartofsky. Totowa: Humana Press

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